What are adeno-associated viruses? To define them simply, they are small viruses that can infect humans and primates. They belong to the same family of parvoviridae viruses that we often vaccinate our dogs and cats against.
Adeno-associated viruses belong to the same family of parvoviridae viruses that we often vaccinate our dogs and cats against. The same family of viruses that bioprocessing scientists find as potential contaminants in cell cultures such as the minute virus of mice (MVM). Critically, while AAV can be found everywhere, there is no evidence to date that they can cause disease – at most, they trigger a very mild immune response.
Simple by nature, AAVs are not enveloped with lipids and are very stable even when they dry out. They have linear, single-stranded DNA (ssDNA) with a genome size of around 4.8 kilobases (kb). Their size is around 20 nm. They cannot replicate themselves but can infect both resting and dividing cells and integrate stably into the human genome. They can replicate only when cells are co-infected with a helper-virus such as an adenovirus or herpes virus.
Suitability for gene therapy
Studies have shown that AAVs alone do not cause any disease and are therefore not pathogenic. When an AAV enters a human cell, it can integrate DNA in chromosome 19 which is stable and persists there in a stable manner, with the virus genome in the cell nucleus being inactive. This can be described as latent infection and can only replicate within human cells with the help of other viruses. These properties make AAV suitable viral vectors for gene therapy to introduce intact genes into human cells in a stable and sufficiently targeted manner, without carrying the risk of causing disease or stimulating a severe immune response. In addition, their genome structure is not very complex, which simplifies experimental manipulation. A wide range of different AAV variants (serotypes) are known today. Their envelope proteins have unique surface structures, which means that they differ in the choice of their target cells (tropism). Since a given vector genome can easily be packaged in the envelope of any AAV serotype, there are hardly any cell types which would not be amenable to the introduction of AAV vector DNA. This versatility and their natural simplicity have made AAVs a valuable asset in the advancement of gene therapies.
Recombinant AAVs to create efficient gene target therapies
AAVs that have genetic material replaced by foreign DNA are referred to as recombinant AAV (rAAV).
Recombinant AAV-derived vectors are infectious, non-replicating particles with parts of AAV DNA that can transfer foreign DNA. Because the virus genome is only 4.8 kb in size, cloning capacity is limited, so most therapeutic genes require a complete replacement of the virus genome. AAV are therefore not suitable for cloning large genes. While this is a limitation, the therapeutic opportunities achievable by the replacement of small genes are significant and do not prevent the successful use of AAVs in gene therapies. A number of ideas to increase gene size are currently being researched, such as the implementation of dual vectors. Here, large genes of interest are split into two separate AAV vectors that, upon co-infection of the same cell, reconstitute the expression of a full-length gene. This has the potential to double the AAV cargo capacity.
With recombinant AAV vectors there remains some risk that the target gene will integrate into chromosome 19 at a random location, for example, in the vicinity of genes that control cell growth. Problems can also arise regarding transgene silencing, deviating transcription regulation, and insertion mutagenesis. Under certain circumstances, this can lead to over-, under-, or incorrect expression of the target gene, the unwanted proliferation of cell clones, or even cancer. Therefore, targeted, and stable integration of the target gene into the recipient genome is an extremely important part of gene therapy and the subject of continued gene targeting research.
In 1998, with specific gene targeting in mind, a working group in Seattle found that recombinant AAVs could integrate into a targeted location in the genome with an accuracy of one percent, if it contained the appropriate homology sequences. In the case of targeted recombinations it was even possible to achieve an accuracy of only ten random integrations – a remarkable success which, surprisingly, did not depend on the cell type or the target sequence. The genome of classic rAAVs consist of single-stranded DNA, with many indications that this plays an important role in gene targeting. However, the exact mechanism and reason for this exceptionally efficient homologous integration is still unknown. One theory suggests this is due to a recombination mechanism in which the ssDNA pairs with the homologous sequences in the genome, and acts as a template for recombination. However, comparable targeted integration events have also been shown with novel rAAV vectors present in the target cell as double-stranded DNA. Realization of the exact integration mechanism of rAAVs clearly remains an exciting goal for future research.
Recombinant AAVs have many advantageous properties
In addition to the high gene targeting efficiency, other properties of rAAVs are also advantageous for targeted gene modification. The different serotypes of AAV make it possible to genetically modify different cell types from different species. This possibility even extends to modifying cells in which the introduction of exogenous DNA by transfection, or via other viral vectors, often proves difficult.
Recombinant AAV vectors are currently being tested in more than 250 clinical studies worldwide as therapeutics for a wide variety of diseases, with some AAV-based drugs already approved (Luxturna, Glybera). Today’s reality is that rAAVs can be developed into life-changing gene-targeted medicines that deliver genuine hope for curing chronic illnesses.
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Glybera is a trademark of uniQure IP B.V. Luxturna is a trademark of Spark Therapeutics, Inc.